The mammalian nervous system comprises a peripheral nervous system (PNS) and a central nervous system (CNS, comprising the brain and spinal cord), and is composed of two principal classes of cells: neurons and glial cells. The glial cells fill the spaces between neurons, nourishing them and modulating their function. Certain glial cells, such as Schwann cells in the PNS and oligodendrocytes in the CNS, also provide a myelin sheath that surrounds neural processes. The myelin sheath enables rapid conduction of impulses along the neuron. In the peripheral nervous system, axons of multiple neurons may bundle together in order to form a nerve fiber. These, in turn, may be combined into fascicles or bundles.
During development, differentiating neurons from the central and peripheral nervous systems send out axons that grow and make contact with specific target cells. In some cases, axons must cover enormous distances; some grow into the periphery, whereas others are confined within the central nervous system. In mammals, this stage of neurogenesis is complete during the embryonic phase of life and neuronal cells do not multiply once they have fully differentiated.
A host of neuropathies have been identified that affect the nervous system. These neuropathies, which may affect neurons themselves or associated glial cells, may result from cellular metabolic dysfunction, infection, exposure to toxic agents, autoimmunity, malnutrition, or ischemia. In some cases, the neuropathy is thought to induce cell death directly. In other cases, the neuropathy may induce sufficient tissue necrosis to stimulate the body""s immune/inflammatory system and the immune response to the initial injury then destroys neural pathways.
Attempts to counteract the effects of acute or neurodegenerative lesions of the brain and/or spinal cord have primarily involved implantation of embryonic neurons in an effort to compensate for lost or deficient neural function. However, human fetal cell transplantation research is severely restricted. Administration of neurotrophic factors such as nerve growth factor and insulin-like growth factor also have been suggested to stimulate neuronal growth within the CNS. See, e.g., Lundborg, Acta Orthop. Scand. 58: 145-169 (1987); U.S. Pat. No. 5,093,317. Administration of neurotrophic factors to the CNS requires bypassing the blood-brain barrier. The barrier may be overcome by direct infusion, or by modifying the molecule to enhance its transport across the barrier, as by chemical modification or conjugation, or by molecule truncation. Schwann cells also have been grafted to a site of a CNS lesion in an attempt to stimulate and maintain growth of damaged neuronal processes. Paino, et al., Exp. Neurology 114: 254-257 (1991).
One type of morphoregulatory molecule associated with neuronal cell growth, differentiation and development is the cell adhesion molecule (xe2x80x9cCAMxe2x80x9d), most notably the nerve cell adhesion molecule (N-CAM). The CAMs are members of the immunoglobulin superfamily. They mediate cell-cell interactions in developing and adult tissues through homophilic binding, i.e., CAM-CAM binding on apposing cells. A number of different CAMs have been identified. Of these, the most thoroughly studied are N-CAM and L-CAM (liver cell adhesion molecules), both of which have been identified on all cells at early stages of development, as well as in different adult tissues. In neural tissue development, N-CAM expression is believed to be important in tissue organization, neuronal migration, nerve-muscle tissue adhesion, retinal formation, synaptogenesis, and neural degeneration. Reduced N-CAM expression also is thought to be associated with nerve dysfunction. For example, expression of at least one form of N-CAM, N-CAM-180, is reduced in a mouse demyelinating mutant. Bhat, Brain Res. 452: 373-377 (1988). Reduced levels of N-CAM also have been associated with normal pressure hydrocephalus, Werdelin, Acta Neurol. Scand. 79: 177-181 (1989), and with type II schizophrenia. Lyons, et al., Biol. Psychiatry 23: 769-775 (1988). In addition, antibodies against N-CAM have been shown to disrupt functional recovery of injured nerves. Remsen, Exp. Neurobiol. 110: 268-273 (1990).
Where the damaged neural pathway results from CNS axonal damage, autologous peripheral nerve grafts have been used to bridge lesions in the central nervous system and to allow axons to make contact with their normal target cell. In contrast to CNS neurons, neurons of the peripheral nervous system can extend new peripheral processes in response to axonal damage. This regenerative property of peripheral axons is thought to be sufficient to allow grafting of these segments to CNS axons. Successful grafting appears to be limited, however, by a number of factors, including the length of the CNS axonal lesion to be bypassed, and the distance of the graft sites from the CNS neuronal cell bodies, with successful grafts typically occurring near the cell body.
No satisfactory method exists to repair the damage caused by neuropathies, such as Parkinson""s disease (parkinsonism). Parkinson""s disease is a syndrome consisting of tremor, rigidity, brady- and hypokinesia, and deficits in equilibrium and posture. The substantia nigra is the principal site of pathology in Parkinson""s disease. Pigmented neurons of the substantia nigra project widely and diffusely to the caudate-putamen (corpus striatum) and are specialized to synthesize and release dopamine. There are approximately 500,000 specialized dopaminergic cells in the pars compacta of the substantia nigra of young adults. Symptoms of parkinsonism emerge when 75-80% of the dopaminergic innervation is destroyed. Patients with Parkinson""s disease respond to dopamine replacement therapy. Unfortunately, the efficacy of dopamine replacement therapy decreases progressively with continued degeneration of the nigrostriatal dopaminergic pathway. Accordingly, there is a need in the art for treatments of neurological disorders, such as Parkinson""s disease, and related deficits in neural function.
The present invention provides methods and compositions for treating Parkinson""s disease, including methods for enhancing the survival of neural cells. In one aspect, the invention features compositions and therapeutic treatment methods comprising administering to a mammal a therapeutically effective amount of a morphogenic protein (xe2x80x9cmorphogenxe2x80x9d), as defined herein, upon diagnosis of Parkinson""s disease, or upon detecting degeneration of the nigrostriatal pathway prior to the appearance of symptoms of parkinsonism (i.e., when less than 75-80% of the dopaminergic innervation is destroyed).
In a preferred embodiment, methods of the invention for treating Parkinson""s disease comprise administering a morphogen comprising a dimeric protein having an amino acid sequence selected from the group consisting of a sequence having 70% homology with the C-terminal seven-cysteine skeleton of human OP-1 (amino acids 330-341 of SEQ ID NO: 2); a sequence having greater than 60% amino acid sequence identity with human OP-1; generic sequence 7 (SEQ ID NO: 4); generic sequence 8 (SEQ ID NO: 5); generic sequence 9 (SEQ ID NO: 6); generic sequence 10 (SEQ ID NO: 7); and OPX (SEQ ID NO: 3); wherein the morphogen stimulates production of N-CAM or L1 isoform by an NG108-15 cell in vitro. The same or similar methods are contemplated to restore neural cell function in a mammal having Parkinson""s disease. Administering one of the aforementioned morphogens also provides a prophylactic function. Such administration has the effect of preserving neural cell function in a mammal having, or at risk of having, Parkinson""s disease. Also according to the invention, morphogen administration preserves the integrity of the nigrostriatal pathway, as well as other pathways, in the brain. Prophylactically, morphogens of the invention also prevent degeneration of the nigrostriatal pathway or loss of functional activity associated with this pathway.
Specifically, methods of the invention for treating (pre- or post-symptomatically) Parkinson""s disease and damage to neural pathways, especially the nigrostriatal pathway, comprise administering a morphogen selected from the group consisting of human OP-1, mouse OP-1, human OP-2, mouse OP-2, 60A, GDF-1, BMP2A, BMP2B, DPP, Vgl, Vgr-1, BMP3, BMP5, and BMP6. Such morphogens are capable of stimulating production of N-CAM or L1 isoform by an NG108-15 cell in vitro.
In a particularly preferred embodiment, the morphogen is a soluble complex, comprising at least one morphogen pro domain, or fragment thereof, non-covalently attached to a mature morphogen.
In another aspect, the invention features compositions and therapeutic treatment methods for maintaining neural pathways, such treatment methods include administering to the mammal, upon injury to a neural pathway or in anticipation of such injury, a compound that stimulates a therapeutically effective concentration of an endogenous morphogen. These compounds are referred to herein as morphogen-stimulating agents, and are understood to include substances which, when administered to a mammal, act on tissue(s) or organ(s) that normally are responsible for, or capable of, producing a morphogen and/or secreting a morphogen, and which cause endogenous level of the morphogen to be altered.
The invention also provides methods for stimulating neurons to maintain their differentiated phenotype, including inducing the redifferentiation of transformed cells of neuronal origin to a morphology characteristic of untransformed neurons. In one embodiment, the invention provides means for stimulating production of cell adhesion molecules, particularly nerve cell adhesion molecules (N-CAM). The invention also provides methods, compositions and devices for stimulating cellular repair of damaged neurons and neural pathways, including regenerating damaged dendrites or axons. In addition, the invention also provides means for evaluating the status of nerve tissue, and for detecting and monitoring neuropathies by monitoring fluctuations in morphogen levels.
In one aspect of the invention, the morphogens described herein are useful in repairing damaged neural pathways of the peripheral nervous system. In particular, morphogens are useful for repairing damaged neural pathways, including transected or otherwise damaged nerve fibers. Specifically, morphogens described herein are capable of stimulating complete axonal nerve regeneration, including vascularization and reformation of the myelin sheath. Preferably, the morphogen is provided to the site of injury in a biocompatible, bioresorbable carrier capable of maintaining the morphogen at the site and, where necessary, means for directing axonal growth from the proximal to the distal ends of a severed neuron. For example, means for directing axonal growth may be required where nerve regeneration is to be induced over an extended distance, such as greater than 10 mm. Many carriers capable of providing these functions are envisioned. For example, useful carriers include substantially insoluble materials or viscous solutions prepared as disclosed herein comprising laminin, hyaluronic acid or collagen, or other suitable synthetic, biocompatible polymeric materials such as polylactic, polyglycolic or polybutyric acids and/or copolymers thereof. A preferred carrier comprises an extracellular matrix composition derived, for example, from mouse sarcoma cells.
In a particularly preferred embodiment, a morphogen is disposed in a nerve guidance channel which spans the distance of the damaged pathway. The channel acts both as a protective covering and a physical means for guiding growth of a neurite. Useful channels comprise a biocompatible membrane, which may be tubular in structure, having a dimension sufficient to span the gap in the nerve to be repaired, and having openings adapted to receive severed nerve ends. The membrane may be made of any biocompatible, nonirritating material, such as silicone or a biocompatible polymer, such as polyethylene or polyethylene vinyl acetate. The casing also may be composed of biocompatible, bioresorbable polymers, including, for example, collagen, hyaluronic acid, polylactic, polybutyric, and polyglycolic acids. In a preferred embodiment, the outer surface of the channel is substantially impermeable.
The morphogen may be disposed in the channel in association with a biocompatible carrier material, or it may be adsorbed to or otherwise associated with the inner surface of the casing, such as is described in U.S. Pat. No. 5,011,486, provided that the morphogen is accessible to the severed nerve ends.
Morphogens described herein are also useful in autologous peripheral nerve segment implants, such as in the repair of damaged or detached retinas, or other damage to the optic nerve.
In another aspect of the invention, morphogens described herein are useful to protect against damage associated with the body""s immune/inflammatory response to an initial injury to nerve tissue. Such a response may follow trauma to nerve tissue, caused, for example, by an autoimmune dysfunction, neoplastic lesion, infection, chemical or mechanical trauma, disease, by interruption of blood flow to the neurons or glial cells, or by other trauma to the nerve or surrounding material. For example, the primary damage resulting from hypoxia or ischemia-reperfusion following occlusion of a neural blood supply, as in an embolic stroke, is believed to be immunologically associated. In addition, at least part of the damage associated with a number of primary brain tumors also appears to be immunologically related. Application of a morphogen, either directly or systemically alleviate and/or inhibit the immunologically related response to a neural injury. Alternatively, administration of an agent capable of stimulating morphogen expression and/or secretion in vivo, preferably at the site of injury, may also be used. Where the injury is to be induced, as during surgery or other aggressive clinical treatment, the morphogen or agent may be provided prior to induction of the injury to provide a neuroprotective effect to the nerve tissue at risk.
Generally, morphogens useful in methods and compositions of the invention are dimeric proteins that induce morphogenesis of one or more eukaryotic (e.g., mammalian) cells, tissues or organs. Tissue morphogenesis includes de novo or regenerative tissue formation, such as occurs in a vertebrate embryo during development. Of particular interest are morphogens that induce tissue-specific morphogenesis at least of bone or neural tissue. As defined herein, a morphogen comprises a pair of polypeptides that, when folded, form a dimeric protein that elicits morphogenetic responses in cells and tissues displaying morphogen-specific receptors. That is, the morphogens generally induce a cascade of events including all of the following in a morphogenically permissive environment: stimulating proliferation of progenitor cells; stimulating the differentiation of progenitor cells; stimulating the proliferation of differentiated cells; and, supporting the growth and maintenance of differentiated cells. xe2x80x9cProgenitorxe2x80x9d cells are uncommitted cells that are competent to differentiate into one or more specific types of differentiated cells, depending on their genomic repertoire and the tissue specificity of the permissive environment in which morphogenesis is induced. An exemplary progenitor cell is a hematopoeitic stem cell, a mesenchymal stem cell, a basement epithelium cell, a neural crest cell, or the like. Further, morphogens can delay or mitigate the onset of senescence- or quiescence-associated loss of phenotype and/or tissue function. Still further, morphogens can stimulate phenotypic expression of a differentiated cell type, including expression of metabolic and/or functional, e.g., secretory, properties thereof. In addition, morphogens can induce redifferentiation of committed cells (e.g., osteoblasts, neuroblasts, or the like) under appropriate conditions. As noted above, morphogens that induce proliferation and/or differentiation at least of bone or neural tissue, and/or support the growth, maintenance and/or functional properties of neural tissue, are of particular interest herein. See, e.g., WO 92/15323, WO 93/04692, WO 94/03200 (providing more detailed disclosures as to the tissue morphogenic properties of these proteins).
As used herein, the terms xe2x80x9cmorphogen,xe2x80x9d xe2x80x9cbone morphogen,xe2x80x9d xe2x80x9cbone morphogenic protein,xe2x80x9d xe2x80x9cBMP,xe2x80x9d xe2x80x9cmorphogenic proteinxe2x80x9d and xe2x80x9cmorphogenetic proteinxe2x80x9d all embrace the class of proteins typified by human osteogenic protein 1 (hOP-1). Nucleotide and amino acid sequences for hOP-1 are provided in SEQ ID NOS: 1 and 2, respectively., For ease of description, hOP-1 is considered a representative morphogen. It will be appreciated that OP-1 is merely representative of the TGF-xcex2 subclass of true tissue morphogens and is not intended to limit the description. Other known and useful morphogens include, but are not limited to, BMP-2, BMP-3, BMP-3b, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-12, BMP-13, BMP-15, GDF-1, GDF-2, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-1, GDF-12, 60A, NODAL, UNIVIN, SCREW, ADMP, and NEURAL, and morphogenically-active amino acid variants of any thereof.
In specific embodiments, useful morphogens include those sharing the conserved seven cysteine skeleton, and sharing at least 70% amino acid sequence homology (similarity), within the C-terminal seven-cysteine skeleton of human OP-I, residues 330-431 of SEQ ID NO: 2 (hereinafter referred to as the xe2x80x9creference sequencexe2x80x9d). In another embodiment, the invention encompasses use of biologically active species (phylogenetic) variants of any of the morphogenic proteins recited herein, including conservative amino acid sequence variants, proteins encoded by degenerate nucleotide sequence variants, and morphogenically-active proteins sharing the conserved seven cysteine skeleton as defined herein and encoded by a DNA sequence to hybridize under standard stringency conditions to a DNA encoding a morphogenic protein disclosed herein, including, without limitation, OP-1 or BMP-2 or BMP-4. Presently, however, the reference sequence is that of residues 330-431 of SEQ ID NO: 2 (OP-1).
In still another embodiment, morphogens useful in methods and compositions of the invention are defined as morphogenically-active proteins having any one of the generic sequences defined herein, including OPX (SEQ ID NO: 3) and Generic Sequences 7 and 8 (SEQ ID NOS: 4 and 5, respectively), or Generic Sequences 9 and 10 (SEQ ID NOS: 6 and 7, respectively). OPX encompasses the observed variation between the known phylogenetic counterparts of the osteogenic OP-1 and OP-2 proteins, and is described by the amino acid sequence presented herein below and in SEQ ID NO: 3. Generic Sequence 9 is a 96 amino acid sequence containing the C-terminal six cysteine skeleton observed in hOP-1 (residues 335-431 of SEQ ID NO: 2) and wherein the remaining residues encompass the observed variation among OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-8, BMP-9, BMP-10, BMP-11, BMP-15, GDF-1, GDF-3, GDF-5, GDF-6, GDF-7, GDF-8, GDF-9, GDF-10, GDF-I 1, 60A, UNIVIN, NODAL, DORSALIN, NEURAL, SCREW and ADMP. That is, each of the non-cysteine residues is independently selected from the corresponding residue in this recited group of known, naturally-sourced proteins. Generic Sequence 10 is a 102 amino acid sequence which includes a five amino acid sequence added to the N-terminus of the Generic Sequence 9 and defines the seven cysteine skeleton observed in hOP-1 (330-431 SEQ ID NO: 2). Generic Sequences 7 and 8 are 96 and 102 amino acid sequences, respectively, containing either the six cysteine skeleton (Generic Sequence 7) or the seven -cysteine skeleton (Generic Sequence 8) defined by hOP-1 and wherein the remaining non-cysteine residues encompass the observed variation among OP-1, OP-2, OP-3, BMP-2, BMP-3, BMP-4, 60A, DPP, Vgl, BMP-5, BMP-6, Vgr-1, and GDF-1.
Of particular interest are morphogens which, when provided to a specific tissue of a mammal, induce tissue-specific morphogenesis or maintain the normal state of differentiation and growth of that tissue. In preferred embodiments, the present morphogens induce the formation of vertebrate (e.g., avian or mammalian) body tissues, such as but not limited to nerve, eye, bone, cartilage, bone marrow, ligament, tooth dentin, periodontium, liver, kidney, lung, heart, or gastrointestinal lining. Preferred methods may be carried out in the context of developing embryonic tissue, or at an aseptic, unscarred wound site in post-embryonic tissue. Methods of identifying such morphogens, or morphogen receptor agonists, are known in the art and include assays for compounds which induce morphogen-mediated responses (e.g., induction of endochondral bone formation, induction of differentiation of metanephric mesenchyme, and the like). In a preferred embodiment, morphogens of the invention, when implanted in a mammal in conjunction with a matrix permissive of bone morphogenesis, are capable of inducing a developmental cascade of cellular and molecular events that culminates in endochondral bone formation. See, U.S. Pat. No. 4,968,590, Sampath, et al., Proc. Natl. Acad. Sci. USA 80: 6591-6595 (1983), the disclosures of which are incorporated by reference herein.
In an alternative preferred embodiment, morphogens of the invention are also capable of stimulating production of cell adhesion molecules, including nerve cell adhesion molecules (N-CAMs). In a preferred embodiment, the present morphogens are capable of stimulating the production of N-CAM in vitro in NG108-15 cells, which are a preferred model system for assessing neuronal differentiation, particularly motor neuron differentiation.
In still other embodiments, an agent which acts as an agonist of a morphogen receptor may be administered instead of the morphogen itself. An xe2x80x9cagonistxe2x80x9d of a receptor is a compound which binds to the receptor, and for which the result of such binding is similar to the result of binding the natural, endogenous ligand of the receptor. That is, the compound must, upon interaction with the receptor, produce the same or substantially similar transmembrane and/or intracellular effects as the endogenous ligand. Thus, an agonist of a morphogen receptor binds to the receptor and such binding has the same or a functionally similar result as morphogen binding (e.g., induction of morphogenesis). The activity or potency of an agonist can be less than that of the natural ligand, in which case the agonist is said to be a xe2x80x9cpartial agonist,xe2x80x9d or it can be equal to or greater than that of the natural ligand, in which case it is said to be a xe2x80x9cfull agonist.xe2x80x9d Thus, for example, a small peptide or other molecule which can mimic the activity of a morphogen in binding to and activating the morphogen""s receptor may be employed as an equivalent of the morphogen. Preferably the agonist is a full agonist, but partial morphogen receptor agonists may also be advantageously employed. Such an agonist may also be referred to as a morphogen xe2x80x9cmimic,xe2x80x9d xe2x80x9cmimetic,xe2x80x9d or xe2x80x9canalog.xe2x80x9d
Morphogen inducers and agonists can be identified by mutation, site-specific mutagenesis, combinatorial chemistry, etc. Such methods are well known in the art. For example, methods of identifying morphogen inducers or agonists of morphogen receptors may be found in U.S. Pat. No. 6,040,431, filed Jun. 7, 1995 and U.S. Pat. No. 5,834,188 filed Jul. 26, 1995, the disclosures of which are incorporated herein by reference. Candidate morphogen inducers and agonists are then tested for their ability to induce endochondral bone formation and preferably, to stimulate N-CAM production in neurons or in a neuronal model system, such as NG108-15 cells. Morphogen inducers and agonists identified according to the present invention are capable of inducing endochondral bone formation when implanted in a mammal in conjunction with a matrix permissive of bone morphogenesis and are capable of stimulating production of N-CAM in vitro.
The preferred methods, material, and examples that will now be described are illustrative only and are not intended to be limiting. Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.